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Robert Schlapak

Publications and source records attributed to Robert Schlapak.

4 recordsLinked to original sources
PubMed2006

RNA expression profiling at the single molecule level.

We developed a microarray platform for PCR amplification-independent expression profiling of minute samples. A novel scanning system combined with specialized biochips enables detection down to individual fluorescent oligonucleotide molecules specifically hybridized to their complementary sequence over the entire biochip surface of cm2 size. A detection limit of 1.3 fM target oligonucleotide concentration--corresponding to only 39,000 molecules in the sample solution--and a dynamic range of 4.7 orders of magnitude have been achieved. The applicability of the system to PCR amplification-independent gene-expression profiling of minute samples was demonstrated by complex hybridization of cDNA derived from the equivalent of only 10(4) cells, which matches results obtained in ensemble studies on large samples. By counting each hybridized molecule on the microarray, the method is insusceptible to gene-specific variations of the labeling, thereby representing a principle advance to conventional ensemble-based microarray analysis.

Cell Line↗
PubMed2006

Glass surfaces grafted with high-density poly(ethylene glycol) as substrates for DNA oligonucleotide microarrays.

Surfaces carrying a dense layer of poly(ethylene glycol) (PEG) were prepared, characterized, and tested as substrates for DNA oligonucleotide microarrays. PEG bis(amine) with a molecular weight of 2000 was grafted onto silanized glass slides bearing aldehyde groups. After grafting, the terminal amino groups of the PEG layer were derivatized with the heterobifunctional cross-linker succinimidyl 4-[p-maleimidophenyl]butyrate to permit the immobilization of thiol-modified DNA oligonucleotides. The stepwise chemical modification was validated with X-ray photoelectron spectroscopy. Goniometry indicated that the PEG grafting procedure reduced surface inhomogeneities present after the silanization step, while atomic force microscopy and ellipsometry confirmed that the PEG layer was dense and monomolecular. Hybridization assays using DNA oligonucleotides and fluorescence imaging showed that PEG grafting improved the yield in hybridization 4-fold compared to non-PEGylated maleimide-derivatized surfaces. In addition, the PEG layer reduced the nonspecific adsorption of DNA by a factor of up to 13, demonstrating that surfaces with a dense PEG layer represent suitable substrates for DNA oligonucleotide microarrays.

Coated Materials, Biocompatible↗
PubMedDate not supplied

Self-assembled monolayers with latent aldehydes for protein immobilization.

Aldehyde functions are widely used for immobilization of biomolecules on glass surfaces but have found little attention for biofunctionalization of self-assembled monolayers (SAMs) on gold, due to interference between thiol and aldehyde functions. This problem was recently solved by synthesis of an alkanethiol that carried a vicinal diol group [Jang et al. (2003) Nano Lett. 3, 691-694]. The latter served as a latent aldehyde function that was unmasked by short exposure of the vicinal diol-terminated SAM to aqueous periodate. However, the synthesis of the new vicinal diol-terminated alkane thiol was time-consuming and had an overall yield of approximately 3.5%. In the present study, a general modular strategy was introduced by which SAM components with vicinal diol functions were rapidly synthesized with high yield: this was accomplished by amide bond formation between a SAM-forming carboxylic acid (exemplified by lipoic acid and 16-mercaptohexadecanoic acid) with 3-aminopropane-1,2-diol, using suitable protecting groups. The disulfide or free thiol group afforded SAM formation on gold and, after periodate oxidation of the vicinal diol functions, proteins were covalently bound via their lysine residues. At 1 mg/mL protein concentration, complete surface coverage was reached within minutes. No further protein was bound by nonspecific adsorption, but cognate proteins were specifically bound with high capacity. Pyrogallol-O-hexadecanoic acid and 10-undecenoic acid were also coupled with 3-aminopropane-1,2-diol by amide bond formation, thereby producing latent aldehyde-containing SAM components for metal oxides and hydrogen-terminated silicon, respectively, to show the general usefulness of the new synthetic design.

Aldehydes↗